Preparation of a tuberculosis test medium by reconstituting a storage stabilized dry powdered Lowenstein-Jensen medium

ABSTRACT

A storage stable Lowenstein-Jensen medium is prepared by spray drying the liquid medium at an outlet temperature of up to 212 DEG  F. to provide a powder having a moisture content of less than 9% by weight. The spray-dried powder is stable at ambient temperature for at least six months and may be easily reconstituted to the fluid Lowenstein-Jensen medium and used in testing for tuberculosis by simple mixing at ambient conditions.

This is a continuation, of application Ser. No. 243,078 filed Apr. 11,1972 and now abandoned.

The present invention relates to a stabilized growth medium fortuberculosis organisms, and more particularly to a stabilized mediumwhich may be stored under ambient conditions for extended periods oftime without degradation or other deterioration of the growth medium.

The detection of the disease tuberculosis has long been performed byculturing a specimen from a suspected host in a medium referred to inthe art as the Lowenstein-Jensen medium, or modifications thereof. Byallowing the sample to incubate for a set period of time in theLowenstein-Jensen medium, the M. tuberculosis organisms will develop tothe substantial exclusion of other organisms and a positive test fortuberculosis is therefore performed. While this test is quite accurateand is widely practiced, the test has a decided disadvantage in that theLowenstein-Jensen medium is not storage stable at ambient temperaturesand even when refrigerated must be used within 30 days of preparation.In view thereof, it has been the practice in the art to use theLowenstein-Jensen medium within a relatively short period afterpreparation. Thus, it is necessary to frequently perform thetime-consuming preparation of fresh medium. The time-consumingpreparation has generally restricted the use of the Lowenstein-Jensenmedium to organizations which would conduct numerous tuberculosis tests.The occasional tester or the smaller institution cannot economicallytest for tuberculosis, due to the difficulty and expense of frequentlypreparing fresh medium.

The basic ingredient in the Lowenstein-Jensen medium is fresh wholeeggs, e.g., eggs no older than three days. The medium must be preparedin a specific manner with specific nutrient salts added to the fresheggs, as is well known in the art. Once the medium has been prepared, itmust be refrigerated and should be used within a relatively short timeof preparation, as discussed above, or the results obtained therewithare questionable. Attempts in the art at extending the useful life of aprepared Lowenstein-Jensen medium have not met with success and the mostcommon expedient is to premix the nutrient salts in a sterile manner andto place the fresh eggs in the salt mixture when the medium is to beprepared.

From the above, it is quite clear that a storage stableLowenstein-Jensen medium would be quite advantageous in the art and thatthe art has long felt the need for a storage stable Lowenstein-Jensenmedium. Accordingly, it is an object of the present invention to providestabilized dry components of a Lowenstein-Jensen medium. It is a furtherobject to provide such stabilized dry components for a Lowenstein-Jensenmedium which can be stored at room temperature for substantial periodsof time without significant deterioration thereof. It is yet anotherobject of the invention to provide a method whereby the stabilized drycomponents for a Lowenstein-Jensen medium may be readily placed in aliquid and thereby made suitable for use in a tuberculosis test. Otherobjects will be apparent from the following disclosure and claims.

It has now been quite unexpectedly discovered that a Lowenstein-Jensenmedium may be stabilized for sustained preservation by spray drying themedium in a conventional spray-dryer at temperatures consistent with notadversely affecting the protein content thereof.

The initial medium prepared for spray-drying will be the same as aconventional Lowenstein-Jensen medium. There are considerable numbers ofspecific procedures for preparing a Lowenstein-Jensen medium or amodification thereof. However, generally, fresh whole eggs areaseptically combined with nutrient salts, stabilizers, inhibitors,thickeners, etc., and mixed until a well-blended mixture is obtained.Thereafter, the mixture is inspissated at less than 80°-90° C for about45 minutes. A commonly used procedure is that of cleaning fresh eggs in5% soda and soap solution, rinsing thoroughly with water, asepticallybreaking the eggs into a sterile flask containing glass beads, addingnutrients, salts etc., shaking to form a uniform mixture or emulsion,placing the mixture in test tubes and autoclaving the tubes in a slantedposition at 80°-90° C for 45 minutes. Sterility is checked by incubatinga test tube at 37° C. The test tubes are stored under refrigeration.

Irrespective of which specific procedure is used, and many are known tothe art, after the Lowenstein-Jensen medium has been prepared, accordingto the present invention, it is introduced into a conventionalspray-drying apparatus. Either a box-type dryer or a tower dryer may beused, since the important parameters are only the temperature of thespray-drying and the resulting moisture content of the powder soobtained. It is important that the medium be dried to a powder at suchtime and temperature combinations as to not adversely affect the proteinof the medium.

As is generally practiced in the spray-drying art, the box or tower(chamber) of the spray dryer is kept at some relatively constant set oftemperatures by means of heated gas passing therethrough. The fluid tobe spray dried is atomized by a nozzle and sprayed into the chamber. Thefluid may be, if desired, heated, e.g., up to temperatures at, orslightly above that of the spray-drying chamber. The temperature of theheated gas introduced into the spray dryer is referred to as the inlettemperature and the temperature of the gas leaving the spray dryer isreferred to as the outlet temperature. In the present case, it has beenfound that the inlet temperature should be no higher than 300° F. Morepreferably, the inlet temperature should be somewhat less than 300° F,and temperatures between 230° F and 280° F., e.g. between 240° F and280° F and particularly preferred. The outlet temperature in all casesmust be less than 212° F and more preferably less than 200° F. Thus,temperatures between 140° F, especially 150° F, and 200° F arepreferred. However, best results are obtained if the outlet temperatureis between 160° F and 190° F and especially between 175° F and 185° F,e.g. 180° F. The fluid to be atomized and spray dried may be at atemperature up to 138° to 140° F, preferably room temperature orslightly higher. The particular apparatus for heating and atomizing thefluid is not critical, nor is the particular procedure for spray dryingcritical. Thus, any conventional spray-drying apparatus and proceduremay be used.

The volume of the spray dryer should be so chosen in combination withthe temperatures involved that the resulting spray-dried powder has amoisture content, on a weight basis, of no greater than 9%, andpreferably less than 7%, e.g. less than about 6%. Generally speaking,moisture contents of between 5% and 7% by weight are suitable for thepresent invention.

The spray dried powder is recovered from the spray dryer in theconventional manner and may then be packaged in any container desired solong as the container prevents ambient contamination. Thus, thespray-dried powder may be simply packaged in plastic or glass jars,flexible film, blister packages, twisted plastic bag packages, foilpackages, wax paper packages and the like. It is not required that thepackage be hermetically sealed or any like special consideration, but itis preferred that ambient contamination be substantially eliminated bythe packaging method. The elimination of ambient contamination is not inconnection with the stabilized Lowenstein-Jensen medium itself, but inconnection with unknown organisms which might be introduced into thereconstituted powder when used in testing for tuberculosis. The driedand packaged powder need not be stored under special temperatures andambient temperatures are quite acceptable therefor. However, extremetemperatures should be avoided for obvious reasons. Accordingly, it ispreferred that the powder not be subjected to freezing temperatures, noris it preferred that the powder be stored at temperatures above 120° F,preferably less than 115° F, although neither of these extremetemperatures will adversely affect the powder unless the powder remainsat those extreme temperatures for prolonged periods. On the other hand,however, the powder may be stored for prolonged periods of time atambient temperatures without significant deterioration thereof. Thus,the powder may be stored at ambient temperatures for up to one year ormore without significant deterioration, although as a precautionarymeasure and as a safety factor, it is preferred that the powder bereconstituted and used within 8 to 10 months of the drying time.

The powder may be reconstituted simply by mixing with water. Of course,it is preferred that the mixing be under sterile conditions and that thewater be both sterilized and distilled. However, inspissation at 80° Cto 90° C for 1 hour may be used if desired. The amount of powder whichis reconstituted in water can, of course, vary considerably, but it ispreferred that the amount which is reconstituted correspond to thatwhich will produce a level of solid as the original freshly preparedLowenstein-Jensen medium. Thus, the solid content of the reconstitutedmedium can vary from as low as 10% to as high as 20% by weight, but thepreferred solid content will be between about 11% and 17%, especiallybetween 13% and 15%. Reconstitution can be accomplished without anyspecial technique or equipment, and simple mixing is quite adequate.Thus, the dried powder and water may be simply hand mixed or mixed witha conventional laboratory stirrer until dissolution thereof takes place.The mixing may be at ambient temperatures or slightly depressed orelevated temperatures and no special consideration in this regard isrequired. Accordingly, temperatures above the freezing point of waterand as high as 120° F can be used for preparing the reconstitutedmixture. However, there is no special advantage obtained by suchvariation in the temperature of the water for reconstitution, andambient water is quite satisfactory.

For purposes of the present specification, the Lowenstein-Jensen mediumis defined as a medium for the growth of tuberculosis organisms(Mycobacterium tuberculosis) and consists essentially of fresh whole eggand nutrient salts. If desired, inhibitors, thickeners, dyes and likenon-essential ingredients may be used. The specific nutrient salts andthe ratio thereof with the fresh whole egg may vary, as is known in theart. Thus, any combination of fresh whole egg and nutrient saltssuitable for growing tuberculosis organisms are intended to be embracedby the disclosure and claims of this specification. A typicalLowenstein-Jensen formulation, however, is as follows: up to about 3%salts, especially up to about 2% salts, and preferably about 1% salts,up to about 10% water for dispersing and dissolving the salts, moregenerally up to about 8% water, and very often about 5 to 6% water.However, additional water may be used if desired for easy dispersementand dissolution of the salts since this only goes to diluting theconcentration of the Lowenstein-Jensen medium. However, too great ofdilution is not preferred, and generally speaking, no more than 15% byweight of the medium will be added water. The remainder of the mediumwill be essentially fresh eggs, but if desired, other ingredients, suchas inhibitors, thickeners, viscosity control agents and the like may beadded. Thus, typically, the medium will also contain up to 7% of potatoflour, especially up to 6% and more generally between 4 and 5%. Also, upto 4%, generally less than 2% of glycerol may be added. An inhibitor ora dye or a material which will function in both manners is alsogenerally added to the medium. Thus, up to about 1% of an inhibitorand/or dye, more generally less than 1%, may be added to the medium.Usually about half a percent of an inhibitor/dye such as Malachite Greenis used in a conventional Lowenstein-Jensen medium. The nutrient saltsused in the medium generally contain magnesium and phosphorus, as wellas potassium and the particular combination of these salts is notnarrowly critical. However, as a typical example, monopotassiumphosphate, magnesium sulfate, and magnesium citrate may be used. Also,typically, asparagine is used in a Lowenstein-Jensen medium. Asparagineis an alkaloid found in the sprouts of many weeds and has the formulaNH₂.CH₂.(COOH).(CH₂ CONH₂). H₂ O. When all of the foregoing ingredientsare used, as is typical in a Lowenstein-Jensen formula, it is convenientto suspend or dissolve the salts in the water to which has been addedglycerol, if glycerol is used in the medium.

The invention will be illustrated by the following examples, but it isto be clearly understood that the invention is not limited thereto andis fully applicable to the foregoing disclosure.

EXAMPLE 1

Lowenstein-Jensen medium was prepared by mixing until uniformly blendedthe ingredients shown in Table 1 below. The procedure for mixing theingredients was as follows: to the water, when used, was added theglycerol, when used, and the salts, when used, were dispersed therein.When water is not used, glycerol is not used and when water is not used,the salts and/or other ingredients are simply dispersed in the fresheggs. Irrespective, after a mixture has been made, it is whipped untilair bubbles are dispersed in the mixture, i.e., a frothy mixture isobtained. After mixing, the ingredients were spray dried in aconventional box-type spray dryer with the inlet-outlet temperature asindicated in the Table. To the spray-dried powder was mixed between 1/2and 1% on a weight basis of finely divided silica of particle sizebetween submicron and micron range (Syloid) to improve flowability ofthe spray-dried powder. Each of the spray-dried compositions obtainedtherefrom were reconstituted with water to prepare a reconstitutedmedium having the same proportions of solids as the medium from whichthe corresponding sample was spray dried. In each case, thereconstituted medium shows all of the properties of the original mediumprior to spray drying.

                                      TABLE I                                     __________________________________________________________________________    Weight % Of            TEST NUMBER                                            Ingredients  1    2    3    4    5    6                                       __________________________________________________________________________    Water        5.45 0    0    0    5.45 5.45                                    Malachite Green                                                                            .059 .059 .059 .059 .059 .059                                    Monopotassium                                                                              .365 .365 .365 0    .365 0                                        phosphate                                                                    Magnesium                                                                      sulfate     .035 .035 .035 0    .035 0                                       Magnesium                                                                      citrate     .087 .087 .087 0    .087 0                                       Asparagine   .525 .525 .525 0    .525 0                                       Potato Flour 4.38 4.38 0    0    0    4.38                                    Glycerol     1.75 0    0    0    0    0                                       Fresh Eggs    BALANCE                                                         SPRAY DRYING                                                                  CONDITIONS                                                                    Outlet Temp. ° F                                                                    180  160  170  185  190  195                                     Inlet Temp. ° F                                                                     280  285  290  275  270  265                                     __________________________________________________________________________

EXAMPLE 2

In order to demonstrate the prolonged storage properties of thespray-dried powder, 8 samples of Test Number 1, as indicated in Table 1,were stored in a tightly capped container for the periods of 1, 2, 3, 4,5, 6, 10 and 12 months. At the end of each storage period, the samplefor that period was reconstituted to provide a medium with the samesolid content as was originally present in the medium prior to spraydrying. Slants of each sample were tested by inoculating withtuberculosis organisms. At the end of an incubation period of 2 weeks,the samples were examined and each showed a positive culture of M.tuberculosis. Accordingly, this example illustrates that the presentspray-dried medium can be stored for prolonged periods of time andthereafter easily reconstituted for ready use in testing fortuberculosis.

From the foregoing, it can be easily appreciated that the presentinvention provides a method of producing a storage stableLowenstein-Jensen medium by preparing that medium and spray-drying thesame in a spray-drying apparatus which has a chamber and a fluid inletequipped with an atomizing nozzle. In the method, the outlet gas fromthe chamber is at temperatures less than 212° F, and the powder producedalways has a moisture content of less than 9%, e.g., about 6.2%.However, preferably the outlet gas is maintained at temperatures between150° F and 200° F and the moisture content is less than 7%. While not soimportant as the outlet temperature, the inlet gas temperature should bebetween 230° F and 300° F, but temperatures between 240° F and 280° Fare preferred. With these latter temperatures, moisture content of theresulting powder of less than 7% are readily obtained.

In the preferred mode of the invention, the gas outlet temperature isbetween 160° F and 190° F, the gas inlet temperature is between 240° Fand 280° F and the moisture content of the resulting powder is less than7% by weight. The process produces an ambient storage stable, dry,powdered and water-reconstitutable Lowenstein-Jensen medium. The powderso produced is composed of spray-dried solids of a Lowenstein-Jensenmedium wherein the solid have a moisture content of less than 9% and arewater soluable (reconstitutable) at ambient temperatures. The powderwill have a moisture content of, preferably, less than 7% and morepreferably less than 6%. The powder will have been produced by aspray-drying process wherein the temperatures (gas inlet and outlettemperatures) are consistent with not causing substantial deteriorationof the protein of the powder, as for example, denaturation, peptizationand like degradation. The dry powder produced is characterized, mainly,by a storage life of at least 6 months under ambient conditions withoutsignificant deterioration of the powder.

It will be readily apparent to those skilled in the art that variousmodifications and changes can easily be accomplished within theframework and disclosure of the foregoing specification. Accordingly,such modifications and changes are intended as incorporated into thespecification and the invention is limited only by the spirit and thescope of the following claims.

What I claim is:
 1. A method for producing a tuberculoses test mediumfrom a reconstituted dry powdered, storage stabilized Lowenstein-Jensenmedium comprising preparing a Lowenstein-Jensen medium which includesfresh whole eggs, nutrient salts and stabilizers, spray drying themedium in a spray dryer at an inlet temperature of between 230° F and280° F and an outlet temperature of between 160° F and 190° F and underdryer conditions consistent with providing the dried powder with amoisture content of less than 9% by weight, recovering and packaging thedried powder, storing the packaged powder under ambient conditions,forming a reconstituted medium having a solids content of between 10-20%by weight from said powder, and inspissating said reconstituted mediumto provide a medium used in testing for tuberculoses.
 2. A methodaccording to claim 1 wherein the said moisture content of said driedpowder is less than 7% by weight.
 3. A method according to claim 1wherein the outlet temperature is between 160° and 190° F, the inlettemperature is between 240° and 280° F and the moisture content is lessthan 7% by weight.
 4. The reconstituted Lowenstein-Jensen tuberculosestest medium produced by the method of claim 1.